Bortezomib suppresses cytokine production by pDCs stimulated with ODN2216 or influenza virus. (A) PBMCs (1 × 10⁶/mL) were cultured without or with the indicated concentrations of bortezomib for 6 hours and washed to remove bortezomib. The cell concentration was then adjusted to 1 × 10⁶/mL and further cultured for 24 hours in the presence of 0.5μM ODN2216 or influenza virus without bortezomib (Wash [+]). Alternatively, PBMCs (1 × 10⁶/mL) were cultured without or with bortezomib for 6 hours, and cultured for 24 hours in the presence of ODN2216 or influenza virus without removing bortezomib (Wash [−]). Concentrations of IFN-α in the supernatants were measured by ELISA. The data are normalized to the value obtained without bortezomib. *P < .05; **P < .01; ***P < .001. The data are shown as means ± SE of 3 independent experiments. P values refer to the comparison between the data obtained without bortezomib and those obtained with each concentration of bortezomib. The means and ranges of absolute concentrations are as follows: ODN2216 (Wash [−]) 4292 pg/mL (1674-9455 pg/mL); influenza (Wash [−]) 1278 pg/mL (1008-1538 pg/mL). (B) Purified pDCs (2 × 10⁵/mL) were cultured without or with the indicated concentrations of bortezomib for 3 hours, and cultured for 24 hours in the presence of 0.5μM ODN2216 without removing bortezomib. Concentrations of IFN-α and IL-6 in the supernatants were measured by ELISA. **P < .01; ***P < .001. The data are shown as means ± SE of 3 independent experiments. The means and ranges of absolute concentrations were as follows: IFN-α, 74 312 pg/mL (48 118-92 759 pg/mL); IL-6, 3904 pg/mL (1970-6343 pg/mL).